Method of preparing and thawing cryopreserved cells

Abstract

A method of preparing and thawing of cryopreserved cells without added DNase and a method of DNase-free isolation of subpopulations of thawed, cryopreserved cells which can be used to prepare and thaw Human Cord Blood cells for immunoaffinity selection and enrichment of CD34+ hematopoietic progenitor cells for expansion and transfusion.

Description

FIELD AND BACKGROUND OF THE INVENTION[0001] The present invention relates to a method of preparing and thawing cryopreserved cells without the use of DNase which is traditionally used for eliminating the formation of cell aggregates, allowing for superior fluid characteristics and efficient isolation of cell subpopulations using liquid separation techniques, and more particularly, to a superior DNase-free method of preparing and thawing frozen Human Cord Blood cells for immunomagnetic selection and enrichment of CD34+ hematopoietic progenitor cells for expansion and transfusion.[0002] Cord Blood-Derived Hematopoietic Progenitor Cells:[0003] Hematopoietic stem cells are the cells in the bone marrow and in the peripheral blood that are able to differentiate into new blood cells. They can be used for the treatment of many congenital and acquired hematological diseases such as, for example, cancer. It is known that cord blood contains large numbers of hematopoietic stem and progenitor c...

AI Technical Summary

Problems solved by technology

Although aggressive anti-cancer treatments systematically kill tumor cells, they also destroy hematopoietic cells, and stem cells among them.

However, with increasing experience it was found that this protocol caused severe aggregation and clumping after thawing, resulting in extensive loss of CD34+ cells during isolation of cells for expansion.

However, upon thawing cells stored by this method, significant cell damage and aggregation of the cells was consistently observed.

Low temperature preservation of cells is generally accompanied by an ice crystallization process, which is disruptive to membranes and subcellular organelles.

However, while the addition of DNase improved the yield of viable CD34+ cells, the immunomagnetic separation of these progenitor cells from cryopreserved cells remained inefficient.

A further disadvantage of the abovementioned DNase method derives from the costly requirement for recombinant human DNase (7).

However, in Demetriou et al, CD34+ cells are not specified, the components of the proposed cryopreservation medium and protocol are complex and differ greatly from the CD34+ cryopreservation methods described herein, and no mention is made of preparation of cells for further selection of subpopulations.